Department of Neuromuscular Research, National Institute of Neuroscience, National Center of Neurology and Psychiatry, Tokyo, Japan.

Abstract

Emery-Dreifuss muscular dystrophy is an inherited muscular disorder clinically characterized by slowly progressive weakness affecting humero-peroneal muscles, early joint contractures, and cardiomyopathy with conduction block. The X-linked recessive form is caused by mutation in the EMD gene encoding an integral protein of the inner nuclear membrane, emerin. In this study, mutant mice lacking emerin were produced by insertion of a neomycin resistance gene into exon 6 of the coding gene. Tissues taken from mutant mice lacked emerin. The mutant mice displayed a normal growth rate indistinguishable from their littermates and were fertile. No marked muscle weakness or joint abnormalities were observed; however, rotarod test revealed altered motor coordination. Electrocardiography showed mild prolongation of atrioventricular conduction time in emerin-lacking male mice older than 40 weeks of age. Electron microscopic analysis of skeletal and cardiac muscles from emerin-lacking mice revealed small vacuoles, which mostly bordered the myonuclei. Our results suggest that emerin deficiency causes minimal motor and cardiac dysfunctions in mice with a structural fragility of myonuclei.

A: Targeting strategy to disrupt Emd. The schema of structure of the wild-type Emd (Wild) and that of the targeting vector (Emd−) is shown at the top. In the mutant allele, bovine growth hormone polyadenylation signals (bGHpA) and PGK-neo cassette (Neor) replace with exon 6. The schema of wild-type (Wild) and mutant (Emd−) proteins are described at the bottom. B: Southern blotting analysis of the XhoI- and EcoRV-digested genomic DNA from the wild-type and the chimeric mouse (Ch). Using a 5′ probe, 17.7-kb and 11.5-kb hybridization bands correspond in size to homologous recombinant and the wild-type allele, respectively. The 3′ probe hybridized ∼13.5-kb wild-type and ∼9.8-kb recombinant alleles.

A: Quantitative RT-PCR. The values of mRNA for emerin are normalized to that of G3PDH. Expression level of emerin mRNA from cardiac and skeletal muscles from hemizygous mutant male mouse (Emd−/y) at 5 weeks of age are normal compared to wild-type male (wild) and heterozygous (Emd+/−) female littermates. B: Double immunostaining of emerin (a, b) and lamin C (c, d) in muscles from wild-type (a, c) and emerin-lacking mice (b, d). Emerin is observed at the nuclear membrane in the wild-type mouse (a) but not detected in the mutant mouse (b). C: Immunoblotting analysis of emerin and lamin C in muscles from wild-type, heterozygous (Emd+/−) and emerin-lacking male (Emdy/−) and female (Emd−/−) mice. A 38-kd emerin band is not detected in the mutant mice, but similar amounts of lamin C protein are noted. Scale bar, 50 μm.

Result of rotarod test. A: The table shows number and ratio of mice that could walk on the rod for 10 minutes. All wild-type mice could complete the task but only 43% of emerin-lacking male mice, 30% of emerin-lacking female mice, and 58% of heterozygous female mice could complete the task. B: Average time that mice were able to remain on the rod was calculated from the same experiment as in A. Although some mutant mice could remain on the rod more than 10 minutes, others dropped within a minute. The mean time that mutant mice could remain on the rotating rod is significantly shorter than wild-type littermates (**P < 0.01, *P < 0.05). Older mutant mice were observed to have worse motor coordination, whereas no difference was seen among the wild-type mice regardless of age.